RESUMEN
Parathyroid hormone-related protein (PTHrP) is a causative factor of humoral hypercalcemia in malignancy. However, it is difficult to explain the mechanism of anorexia/cachexia with PTHrP secretion in detail. Previously, we demonstrated that the expressions of orexigenic peptides increased and anorexigenic peptides decreased under cachectic conditions in rats carrying tumors secreting PTHrP. In this study, we investigated whether such changes in the expression of hypothalamic feeding-regulating peptides can be solely attributed to PTHrP or are a general response under cachectic conditions. Cachectic syndromes were induced in rats by: (i) inoculation of human lung cancer LC-6 cells that secreted PTHrP, (ii) inoculation of human melanoma SEKI cells that secrete not PTHrP but LIF1, (iii) injection of heat-killed Mycobacterium leading to arthritis (AA) and (iv) oral administration of a high dose of 1α,25(OH)(2)D(3) that resulted in hypercalcemia. The LC-6-bearing rats and AA rats were treated with or without anti-PTHrP antibody and indomethacin, respectively, and the expression of the hypothalamic feeding-regulating peptide mRNAs were examined by in situ hybridization histochemistry. The orexigenic peptide mRNAs, such as neuropeptide Y and agouti-related protein, were significantly increased, and that of anorexigenic peptide mRNAs, such as proopiomelanocortin, cocaine- and amphetamine-regulated transcript and corticotropin-releasing hormone were significantly decreased when they developed cachectic syndromes and AA. A high dose of 1α,25(OH)(2)D(3) caused hypercalcemia and body weight loss but did not affect the expression of hypothalamic feeding-regulating peptide mRNAs. The expressions of the hypothalamic feeding-regulating peptides change commonly in different chronic cachectic models without relating to serum calcium levels.
Asunto(s)
Artritis Experimental/metabolismo , Caquexia/metabolismo , Hipotálamo/metabolismo , Leptina/sangre , Factor Inhibidor de Leucemia/metabolismo , Neoplasias Experimentales/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Proteína Relacionada con Agouti/biosíntesis , Animales , Artritis Experimental/complicaciones , Línea Celular Tumoral , Hormona Liberadora de Corticotropina/biosíntesis , Humanos , Hipercalcemia/etiología , Hibridación in Situ , Péptidos y Proteínas de Señalización Intracelular , Masculino , Neoplasias Experimentales/complicaciones , Proteínas del Tejido Nervioso/biosíntesis , Neuropéptido Y/biosíntesis , Neuropéptidos/biosíntesis , Orexinas , Proopiomelanocortina/biosíntesis , ARN Mensajero/análisis , Ratas , Ratas Desnudas , Ratas WistarRESUMEN
A series of 5,5-dimethylthiohydantoin derivatives were synthesized and evaluated for androgen receptor pure antagonistic activities for the treatment of hormone refractory prostate cancer. CH4933468 (32d) with a sulfonamide side chain not only exhibited antagonistic activity with no agonistic activity in the reporter gene assay but also inhibited the growth of bicalutamide-resistant cell lines. This compound also inhibited tumor growth of the LNCaP xenograft in mice dose-dependently.
Asunto(s)
Antagonistas de Andrógenos , Antineoplásicos Hormonales , Ácidos Carboxílicos , Nitrilos/síntesis química , Sulfonamidas/síntesis química , Antagonistas de Andrógenos/síntesis química , Antagonistas de Andrógenos/química , Antagonistas de Andrógenos/farmacología , Animales , Antineoplásicos Hormonales/síntesis química , Antineoplásicos Hormonales/química , Antineoplásicos Hormonales/farmacología , Ácidos Carboxílicos/síntesis química , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Concentración 50 Inhibidora , Masculino , Ratones , Ratones SCID , Estructura Molecular , Nitrilos/química , Nitrilos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Sulfonamidas/química , Sulfonamidas/uso terapéutico , Tiohidantoínas/síntesis química , Tiohidantoínas/química , Tiohidantoínas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 A, and diffracted to 2.0 A resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.
Asunto(s)
Anticuerpos Monoclonales/química , Fragmentos Fab de Inmunoglobulinas/química , Proteína Relacionada con la Hormona Paratiroidea/química , Receptores Acoplados a Proteínas G/química , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Humanos , Pruebas de Neutralización , Estructura Terciaria de ProteínaRESUMEN
Parathyroid hormone-related protein (PTHrP) plays a vital role in the embryonic development of the skeleton and other tissues. When it is produced in excess by cancers it can cause hypercalcemia, and its local production by breast cancer cells has been implicated in the pathogenesis of bone metastasis formation in that disease. Antibodies have been developed that neutralize the action of PTHrP through its receptor, parathyroid hormone receptor 1, without influencing parathyroid hormone action through the same receptor. Such neutralizing antibodies against PTHrP are therapeutically effective in animal models of the humoral hypercalcemia of malignancy and of bone metastasis formation. We have determined the crystal structure of the complex between PTHrP (residues 1-108) and a neutralizing monoclonal anti-PTHrP antibody that reveals the only point of contact is an alpha-helical structure extending from residues 14-29. Another striking feature is that the same residues that interact with the antibody also interact with parathyroid hormone receptor 1, showing that the antibody and the receptor binding site on the hormone closely overlap. The structure explains how the antibody discriminates between the two hormones and provides information that could be used in the development of novel agonists and antagonists of their common receptor.
Asunto(s)
Especificidad de Anticuerpos , AMP Cíclico/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/inmunología , Animales , Sitios de Unión , Neoplasias Óseas , Línea Celular Tumoral , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Pruebas de Neutralización , Osteosarcoma , Conformación Proteica , ARN Mensajero/genética , Ratas , Receptor de Hormona Paratiroídea Tipo 1/química , Receptor de Hormona Paratiroídea Tipo 1/genética , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Propiedades de Superficie , Difracción de Rayos XRESUMEN
We established models of cancer-related anemia in mice from subcutaneous inoculation of two IL-6-producing cancer cell lines, human lung cancer cell line LC-06-JCK and murine colon26 clone 5 colon cancer cells. In both models, elevated levels of IL-6 were detected in sera and hemoglobin levels significantly decreased compared with non-tumor-bearing mice. In the LC-06-JCK model, serum albumin levels also decreased with elevated levels of human IL-6 in sera. On the other hand, serum levels of EPO increased, although anemia developed and did not improve. The development of cancer-related anemia was prevented by the administration of a rat anti-mouse IL-6 receptor antibody, MR16-1, in the LC-06-JCK model. It is therefore suggested that IL-6 causes anemia independent of a reduction in EPO levels. Our preclinical models should be useful for exploring new modalities for the treatment of cancer-related anemia.
Asunto(s)
Anemia/etiología , Eritropoyetina/metabolismo , Interleucina-6/metabolismo , Animales , Anticuerpos Monoclonales/química , Línea Celular Tumoral , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Interleucina-6/química , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , RatasRESUMEN
The 3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethylthiohydantoin derivatives which have carboxy-terminal side chains were synthesized and their agonistic/antagonistic activities against androgen receptor (AR) measured. Among them, compound 13b showed antagonistic activity (IC50=130 nM) with no agonistic activity even at 10000 nM. This compound exhibited significant metabolic stability and oral antiandrogenic activity (ED50=7 mg/kg).
Asunto(s)
Antagonistas de Andrógenos/síntesis química , Antagonistas de Andrógenos/farmacología , Antagonistas de Receptores Androgénicos , Animales , Unión Competitiva/efectos de los fármacos , Células CHO , Cricetinae , Cricetulus , Cristalografía por Rayos X , Genes Reporteros/efectos de los fármacos , Células HeLa , Humanos , Indicadores y Reactivos , Masculino , Ratones , Ratones Endogámicos ICR , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Vesículas Seminales/efectos de los fármacos , Relación Estructura-Actividad , Tiohidantoínas/síntesis química , Tiohidantoínas/farmacologíaRESUMEN
Parathyroid hormone-related peptide (PTHrP) was discovered to be a causative factor of humoral hypercalcemia of malignancy (HHM) . It also causes cachexia including reduced food intake, body weight loss, and decreased locomotor activity by a mechanism that is independent of hypercalcemia and proinflammatory cytokine actions. PTHrP-induced cachexia is not associated with the repression of orexigenic peptides or induction of anorexigenic peptides ; unexpectedly, the expression of orexigenic peptides was increased and that of anorexigenic peptides was decreased in animals that developed PTHrP-induced cachexia. A neutralizing antibody against PTHrP rapidly restored food intake, body weight, and locomotor activity and also normalized the expression of orexigenic and anorexigenic peptides. Thus, PTHrP induces cachexia by mechanisms other than directly affecting the hypothalamic feeding regulated peptides.
Asunto(s)
Regulación del Apetito/genética , Caquexia/etiología , Proteína Relacionada con la Hormona Paratiroidea/fisiología , Animales , Anticuerpos/uso terapéutico , Regulación del Apetito/fisiología , Peso Corporal/genética , Caquexia/fisiopatología , Caquexia/terapia , Humanos , Actividad Motora/genética , Proteína Relacionada con la Hormona Paratiroidea/inmunologíaRESUMEN
Lead optimization of CH4892280 (4), an androgen receptor (AR) pure antagonist, was investigated. Compounds 6 and 7, which have a carboxylic acid at the end of the side chain at the position 7alpha of dihydrotestosterone (DHT), showed partial agonistic activities in reporter gene assay (RGA). Conversion of the steroidal core structure to 17alpha-methyltestosterone gave compound 14, which showed weak pure antagonistic activity. Optimization of the side chain by the insertion of a phenyl ring led to compounds 22 and 28-30, which showed pure antagonistic activities at submicromolar concentrations. The structure-activity relationships were clarified.
Asunto(s)
Antagonistas de Receptores Androgénicos , Receptores Androgénicos/metabolismo , Esteroides/química , Esteroides/farmacología , Animales , Ratones , Modelos Moleculares , Estructura Molecular , Unión Proteica , Receptores Androgénicos/química , Esteroides/síntesis química , Relación Estructura-ActividadRESUMEN
It has been shown that in the mouse colon 26 tumor model, tumors grown in the subcutis (subcutis colon 26) caused early onset of cachectic syndromes, whereas those in the liver (liver colon 26) did not. Both interleukin (IL)-6 and parathyroid hormone-related protein (PTHrP) were involved in the development of cachectic syndromes in this tumor model. However, whether expression of PTHrP and IL-6 is differently regulated in the tumor microenvironment is unclear. In the present study, culturing the colon 26 cells under different conditions in vitro revealed that IL-6 production was increased by monolayer culture under a low-glucose condition but not by spheroid culture. In contrast, PTHrP production was increased by spheroid culture but not by monolayer culture, even under a low-glucose condition. Gene expression profiling revealed that the expression of cyclooxygenase (COX)-2 was up-regulated in both subcutis colon 26 and spheroid cultures, and that COX-2 inhibitor NS-398 suppressed PTHrP production in spheroid cultures. Furthermore, administration of NS-398 decreased the PTHrP level without affecting the tumor growth in mice bearing subcutis colon 26. These results demonstrate that production of PTHrP and IL-6 largely depends on the microenvironments in which tumors are developed or metastasized and that up-regulation of COX-2 in a necrobiotic environment leads to PTHrP production, thereby causing cachectic syndromes.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias del Colon/metabolismo , Ciclooxigenasa 2/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Animales , Becaplermina , Caquexia/etiología , Calcio/metabolismo , Neoplasias del Colon/patología , Inhibidores de la Ciclooxigenasa/farmacología , Perfilación de la Expresión Génica , Glucosa/metabolismo , Humanos , Interleucina-6/metabolismo , Masculino , Ratones , Necrosis , Nitrobencenos/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogénicas c-sis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfonamidas/farmacología , Células Tumorales CultivadasRESUMEN
PURPOSE: Parathyroid hormone-related protein (PTHrP) is a causative factor of humoral hypercalcemia of malignancy (HHM) and concurrent anorexia and wasting. Because changes in the expression of hypothalamic feeding-regulating peptides can directly affect appetites and thereby can cause anorexia and wasting, we addressed whether the cachectic syndromes induced by PTHrP rely on the action of hypothalamic feeding-regulating peptides. EXPERIMENTAL DESIGN: Rats were inoculated with a LC-6 human cancer xenograft that secreted PTHrP, and the mRNA levels of the hypothalamic feeding-regulating peptide genes and serum leptin levels were examined before and after the development of HHM by in situ hybridization histochemistry and ELISA, respectively. Some rats were given the anti-PTHrP antibody. RESULTS AND CONCLUSION: The mRNA levels for the orexigenic peptides, such as the agouti-related protein and the neuropeptide Y in the arcuate nucleus (Arc), were significantly increased after the development of HHM, whereas the mRNA levels for the anorexigenic peptides, such as the proopiomelanocortin in the Arc, the cocaine and amphetamine-regulated transcript in the Arc, and the corticotropin-releasing factor in the paraventricular nucleus, were significantly decreased after the development of HHM. Plasma leptin levels were also reduced in cachectic rats, and the administration of anti-PTHrP antibody to the cachectic rats not only improved the cachectic symptoms but also restored the mRNA levels of these orexigenic and anorexigenic peptides, except for orexin. Thus, PTHrP induces HHM and concurrent cachectic syndromes by mechanisms other than directly modulating the leptin or hypothalamic feeding-regulated peptides.
Asunto(s)
Caquexia/etiología , Regulación Neoplásica de la Expresión Génica , Proteína Relacionada con la Hormona Paratiroidea/fisiología , Péptidos/química , Animales , Línea Celular Tumoral , Humanos , Hipotálamo/metabolismo , Hibridación in Situ , Leptina/sangre , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Neuropéptido Y/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Ratas , Ratas DesnudasRESUMEN
A series of 7alpha-substituted dihydrotestosterone derivatives were synthesized and evaluated for androgen receptor (AR) pure antagonistic activity. From reporter gene assay (RGA), the compound with a side chain containing N-n-butyl-N-methyl amide (19a) showed pure antagonistic activity (IC(50)=340nM, FI(5)>10,000nM), whereas known AR antagonists showed partial agonistic activities. The optimization of 19a led to compound 23 (CH4892280), which showed more potent pure antagonistic activity (IC(50)=190nM, FI(5)>10,000nM). The SARs of tested compounds suggested that the length of the side chain and the substituents on the amide nitrogen are important for pure antagonistic activities.
Asunto(s)
Antagonistas de Receptores Androgénicos , Dihidrotestosterona/análogos & derivados , Dihidrotestosterona/farmacología , Animales , Sitios de Unión , Unión Competitiva/efectos de los fármacos , Células CHO , Cricetinae , Dihidrotestosterona/química , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Células HeLa , Humanos , Estructura Molecular , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
PURPOSE: To clarify the potential of parathyroid hormone-related protein (PTHrP) and interleukin-6 (IL-6) as cachectic factors in a colon 26 model and the effects of capecitabine on cancer cachexia as determined by plasma levels of IL-6 and PTHrP and body weight loss. METHODS: From two colon 26 sublines-cancer cachectic clone20 and non-cachectic clone5 plasma levels of PTHrP protein and mRNA expression levels in tumor tissues were compared. An IL-6 neutralizing antibody, a PTHrP neutralizing antibody, and capecitabine were administered into mice bearing clone20 and their anticachectic effects evaluated. RESULTS: The plasma level of PTHrP protein in mice bearing clone20 was higher than that in mice bearing clone5. The expression level of PTHrP mRNA was 49-fold higher in tumor tissues of clone20 than of clone5, according to GeneChip analysis. PTHrP antibody as well as IL-6 antibody suppressed wasting of the body and gastrocnemius and adipose tissue weights. PTHrP antibody suppressed the induction of hypercalcemia but not hypoglycemia or elevation of IL-6, whereas IL-6 antibody suppressed the induction of hypoglycemia but not hypercalcemia or elevation of PTHrP. Capecitabine, a fluorinated pyrimidine anticancer agent, improved body wasting of mice bearing clone20 at a low dose with no reduction of tumor volume. Furthermore, capecitabine lowered the levels of PTHrP and IL-6 in plasma and suppressed hypoglycemia and hypercalcemia in this model. Capecitabine also showed anticachectic effects on cachexia in a cancer model induced by human cervical cancer cell line Y (also known as Yumoto). CONCLUSIONS: PTHrP and IL-6 were found to be factors in the development of cachexia in a colon 26 cancer model, and capecitabine improved cancer cachexia by suppressing the plasma levels of IL-6 and PTHrP in colon 26 and Y cachectic models.
Asunto(s)
Caquexia/tratamiento farmacológico , Neoplasias del Colon/complicaciones , Desoxicitidina/análogos & derivados , Fluorouracilo/análogos & derivados , Interleucina-6/sangre , Proteína Relacionada con la Hormona Paratiroidea/sangre , Animales , Caquexia/sangre , Caquexia/etiología , Capecitabina , Línea Celular , Desoxicitidina/uso terapéutico , Fluorouracilo/uso terapéutico , Hipercalcemia/prevención & control , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Tumorales Cultivadas , Pérdida de PesoRESUMEN
BACKGROUND: Parathyroid hormone-related protein (PTHrP) has been implicated in bone metastasis. However, the effects on bone metastasis of blocking the PTHrP function have not been tested in the clinic. Here, the effects of a humanized anti-PTHrP monoclonal antibody (mAb) on bone metastasis in a human xenograft model are shown. MATERIALS AND METHODS: Subline MDA-5a, with high bone metastatic activity, was established from the human breast cancer cell line MDA-MB-231. Mice were injected with MDA-5a and an anti-PTHrP monoclonal antibody (mAb) raised against human PTHrP (1-34); bone metastasis was evaluated by X-ray photography. RESULTS: MDA-5a produced elevated levels of PTHrP, Interleukin 8 (IL-8), IL-6 and matrix metalloproteinase 1 (MMP-1) and frequently metastasized to the bone. Administration of the humanized anti-PTHrP mAb significantly suppressed osteolytic bone metastasis of MDA-5a and caused osteogenesis at the sites of metastasis. CONCLUSION: The humanized anti-PTHrP mAb was effective against bone metastasis by inducing osteogenesis and, therefore, will provide a new treatment option for bone metastasis in breast cancer.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Neoplasias Óseas/prevención & control , Neoplasias de la Mama/tratamiento farmacológico , Proteína Relacionada con la Hormona Paratiroidea/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Humanos , Interleucinas/biosíntesis , Interleucinas/genética , Metaloproteinasa 1 de la Matriz/biosíntesis , Metaloproteinasa 1 de la Matriz/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína Relacionada con la Hormona Paratiroidea/biosíntesis , Proteína Relacionada con la Hormona Paratiroidea/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Bisphosphonate and calcitonin lower blood calcium in humoral hypercalcemia of malignancy (HHM) by suppressing osteoclastic bone resorption, but repeated administration of these drugs often leads to relapse. In this study, we examined the roles of parathyroid hormone-related protein (PTHrP) in the development of bisphosphonate- and calcitonin-refractory HHM. EXPERIMENTAL DESIGN: Nude rats bearing the LC-6 JCK tumor xenograft (LC-6 rats) exhibited high bone turnover and HHM. Repeated administration of alendronate induced a sustained suppression of the bone resorption, but it caused only early and transient reduction of the blood calcium levels, leading to unresponsiveness to the drug. Because high blood levels of PTHrP were detected in the LC-6 rats, those that developed alendronate-refractory HHM were treated with an anti-PTHrP antibody. RESULTS: Administration of anti-PTHrP antibody to animals that received repeated administration of alendronate, thereby developing alendronate-refractory HHM, resulted in an increase in fractional excretion of calcium and a marked decrease of blood calcium level. Drug-refractory HHM was also observed in animals that received another osteoclast inhibitor, an eel calcitonin analogue elcatonin. The blood calcium level decreased after the initial administration of elcatonin, but it eventually became elevated during repeated administration. Administration of the anti-PTHrP antibody, but not of alendronate, effectively reduced the blood calcium of the animals that developed elcatonin-refractory HHM. CONCLUSION: High levels of circulating PTHrP and the resulting augmentation of renal calcium reabsorption is one of the major causes of the emergence of osteoclast inhibitor-refractory HHM. Thus, blockage of PTHrP functions by a neutralizing antibody against PTHrP would benefit patients who develop bisphosphonate- or calcitonin-refractory HHM.
Asunto(s)
Alendronato/uso terapéutico , Calcio/metabolismo , Hipercalcemia/etiología , Riñón/metabolismo , Neoplasias Pulmonares/complicaciones , Proteína Relacionada con la Hormona Paratiroidea/fisiología , Alendronato/administración & dosificación , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Resorción Ósea/prevención & control , Calcitonina/administración & dosificación , Calcitonina/análogos & derivados , Calcitonina/uso terapéutico , Calcio/sangre , Calcio/farmacocinética , Línea Celular Tumoral , Difosfonatos/administración & dosificación , Difosfonatos/uso terapéutico , Resistencia a Medicamentos , Humanos , Hipercalcemia/tratamiento farmacológico , Riñón/efectos de los fármacos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Hormona Paratiroidea/sangre , Proteína Relacionada con la Hormona Paratiroidea/sangre , Proteína Relacionada con la Hormona Paratiroidea/inmunología , Ratas , Ratas Endogámicas F344 , Ratas Desnudas , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nude rats bearing the LC-6-JCK human lung cancer xenograft displayed cancer-associated wasting syndrome in addition to humoral hypercalcemia of malignancy. In these rats, not only PTHrP but also several other human proinflammatory cytokines, such as IL-6, leukemia-inducing factor, IL-8, IL-5 and IL-11, were secreted to the bloodstream. Proinflammatory cytokines induce acute-phase reactions, as evidenced by a decrease of serum albumin and an increase in alpha1-acid glycoprotein. Tumor resection abolished the production of proinflammatory cytokines and improved acute-phase reactions, whereas anti-PTHrP antibody affected neither proinflammatory cytokine production nor acute-phase reactions. Nevertheless, tumor resection and administration of anti-PTHrP antibody similarly and markedly attenuated not only hypercalcemia but also loss of fat, muscle and body weight. Body weight gain by anti-PTHrP antibody was associated with increased food consumption; increased body weight from anti-PTHrP antibody was observed when animals were freely fed but not when they were given the same feeding as those that received only vehicle. Furthermore, nude rats bearing LC-6-JCK showed reduced locomotor activity, less eating and drinking and low blood phosphorus; and anti-PTHrP antibody restored them. Although alendronate, a bisphosphonate drug, decreased blood calcium, it affected neither locomotor activity nor serum phosphorus level. These results indicate that PTHrP represses physical activity and energy metabolism independently of hypercalcemia and proinflammatory cytokine actions and that deregulation of such physiologic activities and functions by PTHrP is at least in part involved in PTHrP-induced wasting syndrome.
Asunto(s)
Adenocarcinoma/patología , Carcinoma de Células Escamosas/patología , Metabolismo Energético , Neoplasias Renales/patología , Neoplasias Laríngeas/patología , Neoplasias Pulmonares/patología , Actividad Motora , Neoplasias Pancreáticas/patología , Proteína Relacionada con la Hormona Paratiroidea/fisiología , Síndrome Debilitante/fisiopatología , Adenocarcinoma/veterinaria , Animales , Peso Corporal , Carcinoma de Células Escamosas/veterinaria , Citocinas/sangre , Citocinas/metabolismo , Modelos Animales de Enfermedad , Conducta Alimentaria , Humanos , Hipercalcemia/complicaciones , Hipercalcemia/fisiopatología , Hipercalcemia/veterinaria , Neoplasias Renales/veterinaria , Neoplasias Laríngeas/veterinaria , Neoplasias Pulmonares/veterinaria , Neoplasias Pancreáticas/veterinaria , Ratas , Ratas Desnudas , Trasplante Heterólogo , Síndrome Debilitante/veterinariaRESUMEN
A humanized monoclonal antibody against parathyroid hormone-related protein (PTHrP) was generated from the mouse monoclonal antibody raised against the peptide corresponding to the N-terminal 34 amino acids of the human PTHrP [(PTHrP(1-34)]. The humanized antibody interacted with the PTHrP(1-34) with a kD value of 1.90 x 10(-10) M, and the epitope resides between the amino acids 20 and 30 of the PTHrP. PTHrP(1-34) significantly increased the intracellular cAMP levels in the rat osteosarcoma cells that expressed PTHR1, and the 5 microg/mL or higher concentrations of the humanized antibody almost completely blocked the PTHrP-induced cAMP production even in the presence of 2 microg/mL PTHrP(1-34), demonstrating its ability to fully neutralize PTHrP function. There was no significant difference in the potency of the mouse, chimera, or the humanized antibodies to suppress the PTHrP-induced increase in the intracellular cAMP in ROS cells. Furthermore, at the same doses, the administration of the chimera or the humanized antibody was equally effective in reducing the blood ionized calcium levels of hypercalcemic mice bearing the PAN-7-JCK human pancreatic cancer xenograft or the LC-6-JCK human lung cancer xenograft that secreted PTHrP. Thus, humanized anti-PTHrP may be useful for the treatment of the humoral hypercalcemia of malignancy in humans.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Hipercalcemia/terapia , Proteína Relacionada con la Hormona Paratiroidea/antagonistas & inhibidores , Proteína Relacionada con la Hormona Paratiroidea/inmunología , Fragmentos de Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Línea Celular Tumoral , Humanos , Hipercalcemia/sangre , Hipercalcemia/etiología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/complicaciones , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Datos de Secuencia Molecular , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/complicaciones , Ratas , Ratas Endogámicas F344 , Ratas Desnudas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Parathyroid hormone-related protein (PTHrP) plays a central role in humoral hypercalcemia of malignancy (HHM), which is one of the most frequent paraneoplastic syndromes. PTHrP produced by the tumor acts through a common PTH/PTHrP receptor to promote bone resorption, inhibit calcium excretion from the kidney, and induce hypercalcemia. Patients with HHM often develop cachexia associated with typical symptoms such as anorexia, malaise, nausea, constipation, polyuria, polydipsia, and confusion. The etiology of the cachexia is not fully understood but is thought to be caused by hypercalcemia and various cytokines such as interleukin-6, tumor necrosis factor-alpha, leukemia inhibitory factor, and others. In this study, we investigated the role of PTHrP in hypercalcemia and cachexia in HHM by using humanized anti-PTHrP antibody. A mouse monoclonal antibody that binds to PTHrP amino acid sequence 1-34 and inhibits PTHrP function has been humanized to create a specific and potent agent for the treatment of patients with HHM. The mouse monoclonal antibody has been shown to have antihypercalcemic activity against nude mice bearing human tumors. Because a mouse antibody is highly immunogenic in human patients, the complementarity-determining regions from the mouse antibody were grafted into a human antibody. The resulting humanized antibody specifically recognizes PTHrP(1-34) and neutralizes PTHrP functions in vitro and in vivo. The humanized anti-PTHrP antibody was administered intravenously to HHM model animals bearing tumors such as LC-6 human lung carcinoma. These animals showed symptoms similar to those of patients with HHM (eg, hypercalcemia and cachexia). The humanized anti-PTHrP antibody-treated animals responded with normalization of blood ionized calcium level through an improvement of bone metabolism and calcium excretion. Moreover, the treated animals also showed an improvement in body weight, ultromotivity, metabolic alkalosis, food consumption, water intake, serum phosphorus, and renal function. Consequently, the humanized antibody-treated animals experienced complete resolution of hypercalcemia and cachexia. These results suggest that the humanized antibody would be an effective and beneficial agent for patients with HHM, and that PTHrP is a major pathogenetic factor of hypercalcemia and cachexia in patients with HHM.